JP2015519899A - Ribosomal polynucleotides and related expression systems - Google Patents

Abstract

The present invention provides synthetic or isolated polynucleotides that encode mammalian 18S rRNA that is resistant to pactamycin. Pactamycin resistance is brought about by substitution of one or more single residues in the 18S rRNA sequence, fragments thereof containing the substitution, sequences complementary thereto, or sequences substantially identical thereto. Related systems, methods and kits are also described.

Description

This priority application is based on US Provisional Patent Application No. 61 / 649,453, filed May 21, 2012, and US Provisional Patent Application No. 61 / 778,194, filed March 12, 2013. Alleging the benefit of the priority set forth in Section 119 (e) of the US Patent Act. The priority of the filing date is claimed by this application, the disclosures of these provisional patent applications are hereby incorporated by reference in their entirety.

Insertion of Sequence Listing This application is an electronically equivalent copy of a sequence listing submitted electronically with the present specification via EFS web, and an arrangement submitted electronically with this specification via EFS web. It contains a computer-readable format of the column table, and includes a file called “37651506001WO Sequence_Listing.txt,” which was created on May 20, 2013, of 30,874 bytes. It is hereby incorporated by reference in its entirety.

Report on Government Support The invention described herein was made with grants from the National Institutes of Health (NIH) under grant number GM080771. The US government has certain rights in this invention.

Background Ribosomes are macromolecular structures that catalyze protein synthesis in cells. They are generally composed of two subunits consisting of many proteins and several RNAs. Proteins are involved in subunit structure, stability, and activity. However, it has generally been difficult to assign specific functions to individual ribosomal proteins because many are not essential for ribosome function or have additional extra-ribosomal functions. In contrast, ribosomal RNA (rRNA) is involved in the overall shape of the ribosomal subunit and is involved in enzyme activity that catalyzes protein synthesis. There is a growing body of evidence that rRNA can also affect other aspects of protein synthesis, including mRNA mobilization, regulation of translation efficiency of specific mRNAs, and facilitation of ribosome shunting.

  To study the role of rRNA in ribosome assembly, protein synthesis, and non-canonical aspects of rRNA function, alter rRNA sequences and monitor the activity of modified ribosomal subunits in vivo It needs to be able to do. Analysis of rRNA processing, ribosome accumulation, and higher eukaryotic rRNA function has been hampered by the absence of an expression system that allows the rRNA to be modified and then functionally studied.

  There is a strong need in the art for an effective system for expressing modified rRNA and performing functional analysis, particularly in mammalian systems.

SUMMARY In one aspect, a synthetic or isolated polynucleotide encoding a mammalian 18S rRNA that is resistant to pactamycin is provided. In these polynucleotides, pactamycin resistance is provided by one or more single base substitutions in the 18S rRNA sequence. Also provided are fragments of such polynucleotides having the substitution, complementary sequences, and substantially identical sequences. In certain embodiments, the single base substitution in the polynucleotide providing pactamicin resistance is a position corresponding to G963, A964, C1065 or C1066 of mouse 18S rRNA (SEQ ID NO: 23). In certain preferred embodiments, the single base substitution is at a position corresponding to position G963 of SEQ ID NO: 23. Some of these polynucleotides contain a single base substitution corresponding to the G963A substitution in SEQ ID NO: 23.

  In certain embodiments, the polynucleotide encodes a pactamicin resistant human 18S rRNA or mouse 18S rRNA. In certain embodiments, the polynucleotide comprises SEQ ID NO: 23, which does not have a G963A substitution. In certain related aspects, mammalian 18S rRNA molecules encoded by synthetic or isolated polynucleotides are provided. In another aspect, a vector containing a polynucleotide encoding a pactamycin resistant 18S rRNA is provided. In some of these vectors, the polynucleotide may further comprise 5'ETS (external transcription spacer region) and ITS1 (internal transcription spacer region) of the rDNA sequence. In certain embodiments, the vector further comprises a pol-I promoter or a cytomegalovirus (CMV) promoter. In certain embodiments, the vector further comprises a 3'ETS or SV40 poly (A) signal. In another related aspect, a host cell comprising an expression vector is described herein.

  In another aspect, a method for identifying mutations in 18S rRNA that alter ribosome function is provided. The method comprises (a) introducing additional mutations into a synthetic polynucleotide encoding a mammalian 18S rRNA that is resistant to pactamycin (wherein pactamycin resistance is defined by one or more of the 18S rRNA sequences). Provided by a single base substitution), (b) expressing the synthetic polynucleotide with the further mutation in the presence of pactamycin in a host cell, and (c) expressing the synthetic polynucleotide without the further mutation. It involves detecting changes in the ribosome of the host cell as compared to the control cell. These steps allow the additional mutation to be identified as one modified ribosome function. Typically, the synthetic polynucleotide is present in an expression vector that is introduced into the host cell. In certain embodiments, the pactamycin resistant 18S rRNA has a single base substitution at a position corresponding to G963, A964, C1065 or C1066 of mouse 18S rRNA (SEQ ID NO: 23). For example, the 18S rRNA can have a substitution corresponding to the G963A substitution in SEQ ID NO: 23. One method uses a synthetic polynucleotide comprising SEQ ID NO: 23 without the G963A substitution.

  In another aspect, a method for producing ribosomes having enhanced translation efficiency in mammalian cells is provided. These methods (a) introduce additional mutations into a synthetic polynucleotide encoding a pactamicin resistant mammalian 18S rRNA (where pactamicin resistance is one or more single base substitutions in the 18S rRNA sequence). Provided by (b), (b) introducing a synthetic polynucleotide having the further mutation into a host mammalian cell, (c) culturing the cell in the presence of pactamycin, and (d) the further mutation. Involving detecting enhanced translational efficiency in the cell as compared to a control cell expressing a synthetic polynucleotide that has not. By these methods, ribosomes having enhanced translation efficiency can be obtained. Typically, the synthetic polynucleotide used in these methods is present in an expression vector that is introduced into the host cell. In certain embodiments, the pactamycin resistant 18S rRNA has a single base substitution at a position corresponding to G963, A964, C1065 or C1066 of mouse 18S rRNA (SEQ ID NO: 23). Some of the methods use 18S rRNA encoding a synthetic polynucleotide having a single base substitution corresponding to the G963A substitution in SEQ ID NO: 23. For example, the method can utilize a synthetic polynucleotide comprising SEQ ID NO: 23 without the G963A substitution. In various embodiments, translation efficiency can be determined by measuring the level of a particular polypeptide in a host cell.

  In another aspect, there is provided a kit comprising an expression vector comprising a synthetic polynucleotide encoding a mammalian 18S rRNA that is resistant to pactamycin as described herein (wherein pactamycin resistance is 18S rRNA). Provided by one or more single base substitutions in the sequence). In one embodiment, the kit further comprises a cell comprising the expression vector.

  In another aspect, a method of preferentially translating recombinant mRNA, the method comprising expressing in a mammalian cell a polynucleotide encoding a mammalian 18S rRNA that is resistant to pactamycin ( Here, the polynucleotide encoding 18S rRNA has been further modified by introducing one or more additional mutations into the 18S rRNA encoded by the polynucleotide. The method provides preferential binding of the recombinant mRNAs of the present invention.), The method provides the cells with the recombinant mRNA and is sufficient to reduce or eliminate protein synthesis from the cell's endogenous 40S ribosomal subunit. Exposure to the amount of pactamycin, and hence protein synthesis in the cell Limited to 40S ribosomal subunit containing a 18S rRNA encoded by nucleotides, and further comprising recombinant mRNA is preferentially translated.

  A further understanding of the nature and advantages of the expression systems, methods and kits described herein may become apparent by reference to the remaining portions of the specification and the claims.

FIGS. 1 a-1 c illustrate an rRNA transcription processing pathway and an rRNA expression system according to one embodiment. (A) Schematic representation of rRNA expression platform. The black box shows cells and contains polysomes containing mRNA shown in black lines and ribosomal subunits shown in circles (large circle with 60S subunit; small circle with 40S subunit). The darker 40S subunit is an antibiotic sensitive endogenous subunit and the white 40S subunit contains synthetic 18S rRNA and is antibiotic resistant. Different colors are used to indicate that the subunits are physically distinguishable. Cell penetrating antibiotics bind to the endogenous 40S subunit and inhibit its activity. The synthetic 40S subunit is unaffected and can be translated under these conditions. (B) Primary rRNA transcript of mouse 47S. 47S rRNA is illustrated as a horizontal line. Various sections show the encoded rRNA. 18S rRNA is the RNA component of the 40S ribosomal subunit, and 28S and 5.8S rRNA are the RNA components of the 60S subunit. The black line indicates the external transcription spacer region (5'ETS and 3'ETS) and the internal transcription spacer region (ITS1 and ITS2). The cleavage site in the transcription spacer is indicated by a vertical line and a label. (C) rRNA transcription processing pathway. The processing of transcript precursors is associated with numerous proteins and RNA factors and has been extensively studied in various organisms. Mouse 47S rRNA precursor is transcribed by RNA polymerase I in the nucleus and subsequently processed by two possible pathways. Cleavage proceeds in the direction from 47S rRNA to 18S, 5.8S and 28S rRNA; 45S rRNA can be processed into 18S rRNA by two pathways as described, where various intermediate products are generated. Is done. The major cleavage products resulting from processing sites and 18S rRNA maturation are indicated at each cleavage step. Processing of 5.8S and 28S rRNA involves additional cleavage steps and intermediate products, which have not been described. Figures 2a-2c show the processing analysis of rRNA constructs. (A) p18S. 1-S. Schematic representation of RNA expressed from 6 constructs. The construct contains either the pol-I promoter and 3'ETS, or the CMV promoter and SV40 poly (A) signal. 5'ETS and ITS1 are indicated by thick black lines, 18S rRNA is indicated by yellow bars, and the deletion spacer sequence is indicated by thin dotted lines. The cleavage site is indicated. (B) Northern blot analysis of 18S rRNA processing. N2a cells were transfected with the described construct and RNA was analyzed by the Northern blotting method described in the method (Example 7). The nucleotide site of the single stranded RNA size ladder is shown on the left side of the blot. The blot includes the following controls: A: 100 ng of the 18S WT transcript, B: 100 ng of the tagged transcript, C: untagged p18S. 2 μg total RNA from N2a cells transfected with 1 (Pol-1), D: 2 μg total RNA from N2a cells transfected with mock, E: 2 μg total RNA from N2a cells. Synthetic rRNA was detected by hybridization to a tag sequence inserted using an α-tag probe. The upper band (asterisk) corresponds to the full-length and partially processed transcript. The location of mature 18S rRNA is indicated. (C) p18S. Time course of 18S rRNA accumulation from one construct (Pol-1). For these experiments, N2a cells were transfected and RNA was collected at various times after transfection as described. The controls are the same as in (b).

Figures 3a-3c show an analysis of the 5'ETS sequence contribution to 18S rRNA processing. (A) A schematic representation of the construct shown in FIG. The construct contains a pol-I promoter and 3′ETS. (B) Northern blot analysis of 18S rRNA processing. N2a cells were transfected with the described construct and RNA was analyzed by the Northern blotting method described in the method. Synthetic rRNA was detected by hybridization to a tag sequence inserted using an α-tag probe. Asterisks indicate full-length and partially processed transcripts. The position of mature 18S rRNA is indicated. The controls for this blot are as described in FIG. (C) Top : U3 snoRNA 5 ′ hinge region (SEQ ID NO: 25), p18S. 1 (SEQ ID NO: 26), p18S. 8Δ (SEQ ID NO: 27), and p18S. The figure which shows the comparison with 8m (sequence number: 28); p18S. The complementary sequence that matches 1 is highlighted. Middle : Northern blotting showing synthetic rRNA expression from N2a cells transfected with the described construct. This blot was hybridized using an α-tag probe as in panel (b). The controls for this blot are as described in FIG. Figures 4a-4d show analysis of pactamycin resistant mutants in N2a cells. (A) Protein expression in cells expressing wild type or mutant 18S rRNA constructs. Cells were transfected with constructs expressing untransfected (NT) or wild type (WT) 18S rRNA or 18S rRNA containing the indicated point mutations. Cells were then labeled with ³⁵ S pulse in the absence or presence of the indicated 100 ng / ml pactamycin as described in the method, cells were lysed, and equal amounts of cell lysate were analyzed by SDS-PAGE. A typical autoradiogram is shown. (B) Relative luciferase activity in cell lysates from cells transfected with wild type or mutant 18S rRNA and pGL4.13 (Promega). N2a cells were co-transfected with the indicated 18S rRNA construct and firefly luciferase construct (pGL4.13) and luciferase expression was monitored from cell lysates. Luciferase activity is compared to a lysate obtained from cells transfected with the WT construct (denoted 1.0). Details of co-transfection and assay are described in Example 7. (C) Quantification of synthetic rRNA levels by Northern blotting for cells transfected with WT or mutant rRNA constructs. Synthetic rRNA was detected by hybridization using an α-tag probe. The signal is quantified using a Molecular Dynamics phosphor imager and is shown relative to WT, with WT being 100%. (D) Cells were transfected with either wild type or G693A mutation containing 18S rRNA constructs and incubated with various amounts of pactamycin as indicated. Cells were labeled with ³⁵ S pulse and cell lysates were analyzed by SDS-PAGE. A typical autoradiogram is shown. In panels (c) and (d), error bars represent the standard deviation from three independent experiments.

Figures 5a-5e show the sucrose density gradient distribution of ribosomal subunits containing synthetic 18S rRNA. (A) ddCTP primer extension assay. The wild-type partial sequence (SEQ ID NO: 29) and mutant 18S rRNA are shown. The position of the pactamycin resistance mutation (G693A) located at nucleotide 963 of mouse 18S rRNA is highlighted (SEQ ID NO: 30). The sequence of oligonucleotide primer 693RT (SEQ ID NO: 31) is shown aligned to complementarily match 18S rRNA. Primer extension reactions performed in the presence of ddCTP can be completed with the first G located upstream of the primer. This can result in a 4 nt-extension product (SEQ ID NO: 32) from the wild-type 18S rRNA template and a 6 nt-extension product (SEQ ID NO: 33) from the G693A mutant 18S rRNA. In each case, the extended nucleotide is indicated at the 3 ′ end. (B) Top : p18S. Lysates from cycloheximide treated N2a cells transfected with 1 (G693A) were fractionated in a 10-50% (w / v) linear sucrose gradient. Peaks (left to right) indicate 40S ribosomal subunit, 60S ribosomal subunit, 80S single ribosome, and polysome. Fractions collected for RNA analysis (A-H) are shown. Bottom : RNA prepared from fractions A-H was visualized on an agarose gel stained with ethidium bromide. 28S and 18S rRNA are indicated. (C) Top : Ribosomes dissociated by EDTA were fractionated in a 10-35% (w / v) linear sucrose gradient. Peaks (from left to right) indicate 40S and 60S ribosomal subunits. Middle : RNA analysis as described in panel (b). (D) PAGE analysis of RNA prepared from the fraction of sucrose gradient in panel (b), extended with ddCTP primer using ³³ P-labeled oligonucleotide primer 693RT. Sequencing reactions (lanes c, t, a, g) used the same primers and total RNA as template. The upper band arises from synthetic 18S rRNA (Pact ^r ) and the lower band comes from endogenous 18S rRNA (WT). Lanes 1-3 are controls. Control 1 is an equimolar mixture of in vitro transcripts that contain or lack pactamycin resistance mutations. Control 2 is total RNA from untransfected N2a cells containing only wild type 18S rRNA. Control 3 is a control without a template. The concentration of primer extension products from the various fractions is quantified from the phosphorimager exposure and the ratio of the two bands (WT: Pact ^r ) is shown inserted. (E) ddCTP primer extension from the gradient fraction of panel (c). Figures 6a-6b show an analysis of the function of synthetic 18S rRNA containing deletions in 5'ETS or ITS1. (A) Reporter assay of N2a cells co-transfected with monocistronic reporter constructs expressing optimized firefly luciferase (Fluc2) and various 18S rRNA constructs described above. A schematic of the monocistronic construct is shown above. The synthetic rRNA used in each experiment is shown below the graph: p18S. 1 contains the full length 5'ETS and ITS1, p18S. 7 contains a deletion of the 3 'region of 5'ETS, and p18S. 2 lacks ITS1. Luciferase activity was determined as described in the method. The results of various transfections are described as Fluc2 Fold Induction, which is luciferase activity that exceeds the background obtained with wild-type (pactamicin sensitive) ribosomes (p18S.1). (B) Reporter assay of N2a cells transfected with dicistronic reporter constructs expressing Fluc2 and optimized human Renilla luciferase (hRen). The control construct contained multiple cloning sites (MCS) in the intercistronic region; other constructs contained either EMCV or PV IRES. The 18S rRNA construct and reporter construct used for each experiment are shown below the graph. Results are reported as the ratio of hRen to Fluc2 normalized to MCS constructs without IRES activity. Error bars indicate standard deviation from three independent experiments.

Figures 7a-7e show analysis of 18S rRNA mutations. (A) A schematic representation of the construct. The construct contains a pol-I promoter and 3'ETS. The northern blotting method in panels be includes RNA from N2a cells transfected with various 18S rRNA constructs and RNA from untransfected (control) cells as described above. (B) Northern blot analysis of synthetic 18S rRNA using α-tag probe. (C) Northern blot from panel b was reprobed with α-18S rRNA probe to detect endogenous 18S rRNA in the same sample. (D) Northern blot analysis of 5'ETS-containing pre-rRNA using an α-5'ETS probe that hybridizes to the sequence located 5 'immediately after site 1. (E) Northern blot analysis of ITS1-containing pre-rRNA using an α-ITS1 probe that hybridizes to the sequence located just 3 'of site 2. The sizes in panels d and e are shown for known pre-rRNA, which is consistent with that shown in FIG. Figures 8a-8c show sedimentation analysis of synthetic rRNA species. (A) Northern blot analysis of total RNA (total) and RNA from cell fractions separated by centrifugation into S100 and P100 fractions. RNA is not transfected (N2a) or the p18S. 1 (pol-1) or p18S. Derived from cells transfected with 1 (CMV) construct. In vitro transcripts of 18S rRNA containing wild type 18S rRNA (wt) and hybridization tag (tag) were electrophoresed simultaneously as hybridization and size controls. The blot was hybridized using an α-18S rRNA probe. The right image shows a portion of the left image, indicated by an asterisk, with contrast adjusted to clarify the band, which corresponds to the full-length and partially processed transcript. (B) The blot was hybridized using an α-tag probe. The right image corresponds to a part of the left image with contrast adjusted. Background hybridization using endogenous rRNA α-tag probes is seen in N2a samples. (C) Cells were either ITS1 or p18S. 2 (pol-I) or any of the p18S. Transfected with 1 (pol-I). The blot was hybridized using an α-tag probe. FIG. 9 shows analysis of mature 18S rRNA product from diluted plasmid transfection. Northern blot analysis of synthetic 18S rRNA from cells transfected with various plasmid dilutions. Three vectors from the mature 40S subunit were tested: p18S.1 containing the full-length 5'ETS and ITS1 sequences. P18S.1 containing a deletion in 1,5'ETS. 7, and p18S. 2. Northern blotting is probed with an α-18S tag and the major band corresponds to mature 18S rRNA. Transfection was performed using pBS KS as a filler plasmid to maintain transfection efficiency. Synthetic 18S rRNA levels were determined using p18S. 1 μg of plasmid / transfection per construct is shown on an arbitrary scale normalized to 100.

Detailed description Overview Described herein are mouse 18S rRNA expression systems and methods of use. This rRNA is the RNA component of the small molecule (40S) ribosomal subunit and is transcribed simultaneously as part of a larger transcription precursor with the 5.8S and 28S rRNA components of the large molecule (60S) subunit. (FIG. 1b). Sequence tags were introduced into 18S rRNA that can be detected by hybridization to allow for experiments on transcription, processing, and subcellular distribution of 40S subunits containing synthetic rRNA. To monitor translation of the ribosomal subunit, mutations that confer resistance to inhibition by the antibiotic pactamycin were identified in 18S rRNA. Pactamycin is thought to inhibit translation by binding to the E site in the 40S ribosomal subunit and inhibiting migration of the mRNA-tRNA complex through the ribosome during elongation. Thus, by expression of 18S rRNA with a pactamycin resistance mutation in the cell, the drug specifically inhibits translation from the endogenous 40S subunit of the cell and monitors translation from a subunit containing the pactamycin resistance mutation. Can be used for

  In the present invention, a mammalian 18S rRNA expression system is provided. The expression system provides a sequence tag inserted into the expansion segment 3 of mouse 18S rRNA to monitor expression and cleavage by hybridization. The expression system typically provides pactamycin resistance, allowing functional analysis of 40S ribosomal subunits, including synthetic 18S rRNA, by selectively inhibiting translation from endogenous (pactamicin sensitive) subunits. Single base substitutions in the region coding for the rRNA can also be provided. With such an expression system, the rRNA construct is suitably expressed in the transfected cells, properly processed, taken up by approximately 15% of the 40S subunit, and functions normally based on various criteria. Can be shown.

  Correct processing of the modified 18S rRNA in the expression systems described herein can provide a method for testing the effect of mutations or natural mutations in the 18S rRNA sequence on rRNA processing and ribosome function, and transcripts. It can also be used to investigate the importance of 18S rRNA flanking sequences in the precursor. For example, as detailed in the following examples, for example, the effects of 5 'external transcription spacer (ETS) and first internal transcription spacer (ITS1) in an 18S rRNA expression system were analyzed. Specifically, deletion analysis supports the need for a binding site for U3 snoRNA, but a large segment of the 5 ′ external transcription spacer and the entire first internal transcription spacer (both adjacent to the 18S rRNA). Showed that it is not required for translation from or accumulation of mature subunits.

  By these studies, the present invention provides synthetic polynucleotides and related expression vectors useful for expressing mammalian 18S rRNA and analyzing ribosome function. The invention also provides a method for identifying mutations in rRNA or ribosomal proteins that cause abnormal ribosome function. Furthermore, the present invention provides methods for modifying ribosomal rRNA sequences to produce ribosomes having altered properties, eg, enhanced translational activity. The objects of the invention described herein find various applications in basic research and synthetic biology.

  It should be understood that the objectives described herein should not be limited to specific methods, protocols, and reagents, which are described as being capable of being modified. Unless otherwise noted, the practice of the methods described uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology. For example, exemplary methods are described in the following literature: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (3rd ed., 2001); Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss, Inc. (4th ed., 2000); and Weissbach & Weissbach, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 42 1 -463, 1988.

II. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following references provide those skilled in the art with many general definitions of terms used herein. Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1st ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000 ); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rd ed., 2002) ; Dictionary of Chemistry, Hunt (Ed.), Routledge (1st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds. ), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4th ed., 2000). Some further clarification of these terms is provided herein so that they apply specifically herein.

  As used herein, the singular forms “a”, “an”, and “the” include the plural unless specifically stated otherwise. Thus, for example, “a cell” includes a plurality of such cells, and “a protein” includes one or more proteins and equivalents known to those of skill in the art.

  The eukaryotic ribosome is 80S and consists of a small subunit (40S) and a large subunit (60S). Its 40S subunit has 18S rNA (1900 nucleotides) and about 33 proteins. The large subunit consists of 5S RNA (approximately 120 nucleotides), 28S RNA (approximately 4700 nucleotides), 5.8S RNA (approximately 160 nucleotides) subunits and about 49 proteins. Ribosomes synthesize proteins based on information encoded in mRNA. During this process, both the supplied amino acid and the nascent peptide are bound to the tRNA molecule. Ribosomes contain three RNA binding sites, referred to as A, P, and E, respectively. The A site binds aminoacyl-tRNA (tRNA bound to an amino acid). The P site binds peptidyl-tRNA (tRNA bound to the peptide being synthesized). The E site then binds free tRNA before being taken out of the ribosome. Usually, protein synthesis begins with an initiation codon AUG near the 5 'end of the mRNA. The translation initiation codon of mRNA first binds to the P site of the ribosome.

  Ribosomal DNA (rDNA) consists of non-transcribed spacer (NTS), 5 ′ external transcription spacer (5′ETS), 18S, internal transcription spacer 1 (ITS1), 5.8S, internal transcription spacer 2 (ITS2), 28S, and It consists of a tandem repeat sequence of an operon, which is a transcription unit, consisting of a 3 ′ external transcription spacer (3′ETS) sequence. The rDNA is first transcribed into a polycistronic rRNA transcription precursor. During rRNA maturation, ETS and ITS fragments are removed and rapidly degraded as non-functional mature byproducts. The 5 'external transcription spacer (5'ETS) is essential for 18S rRNA formation, which is the longest untranslated region of ribosomal RNA transcription in higher eukaryotes.

  The term “polynucleotide of interest” (or “gene of interest” or “target gene”) refers to a cistron, open reading frame (ORF), or polypeptide or protein product (“polypeptide of interest” or “target polypeptide”. ") Is intended to include the polynucleotide sequence encoding. For enhanced expression of the polypeptide of interest, the polynucleotide of interest encoding the polypeptide can be introduced into a host cell carrying the modified 18S rRNA expression system described herein. In particular, 18S rRNA is modified (eg, random mutation or targeted mutation) to identify altered 18S rRNA that results in enhanced translation of the polypeptide of interest. For expression of the polypeptide, the polynucleotide of interest is typically present in an expression vector further comprising suitable transcription regulatory elements (eg, promoter sequences) operably linked to the coding sequence. According to certain experimental methods, various polypeptides of interest are suitable for enhanced expression with the methods described herein, such as therapeutic proteins, nutritional proteins and industrially useful proteins.

  As used herein, the term “endogenous” refers to a polynucleotide or polypeptide that is normally found in a wild-type host, while the term “exogenous” is a polynucleotide or polypeptide that is not normally found in a wild-type host. Means.

  “Host cell” means a living cell into which a heterologous polynucleotide sequence has been or has been introduced. A living cell includes both cultured cells and cells in living organisms. Methods for introducing heterologous polynucleotide sequences into cells are known and include, for example, transfection, electroporation, calcium phosphate precipitation, microinjection, transformation, and viral infection. Often, the heterologous polynucleotide sequence introduced into the cell is a replicable expression vector or cloning vector. In certain embodiments, a host cell can be designed to contain a desired gene on its chromosome or on its genome. Many host cells (eg, CHO cells) that can be used as hosts are known in the art. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (3rd ed., 2001); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). In certain preferred embodiments, the host cell is a mammalian cell.

  The terms “nucleotide sequence”, “nucleic acid sequence”, “nucleic acid” or “polynucleotide sequence” refer to either single-stranded or double-stranded deoxyribonucleotides or ribonucleotide polymers and, unless otherwise specified, are naturally occurring. Also included are known analogs of natural nucleotides that hybridize to nucleic acids as well as nucleotides generated in Nucleic acid sequences can be, for example, eukaryotic sequences, eukaryotic mRNA sequences, cDNA sequences from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA (eg, mammalian DNA), and synthetic DNA or RNA sequences. However, it is not limited to these.

  The terms “operably linked” or “operably linked” are functional linkages between genetic elements that link them in a manner that allows their normal function to be performed. Means a bond. For example, a gene is operably linked to a promoter when its transcription is under the control of the promoter, and the produced transcript is normally translated into a protein normally encoded by the gene. Similarly, a translational enhancer element is operably linked to a gene of interest when it allows for up-regulated translation of mRNA transcribed from the gene.

  Screening generally first determines which cells express or do not express screening markers, or have or do not have functional or phenotypic characteristics, and then the desired characteristics It is a two-step method for physically separating cells having Selection is a screening form in which identification and physical separation are achieved simultaneously by the expression of selectable markers and / or detection of functional properties / phenotypic characteristics. For example, under certain genetic conditions, selection allows cells that express the marker to survive while the other cells die, and vice versa. As used herein, screening and selection produces one or more candidate variants (eg, a library thereof) of a reference molecule (eg, 18S rDNA), and then from the candidate variants, the desired structure Or means the step of identifying one or more specific variants that have a function (eg, enhanced translation efficiency).

  A “substantially identical” nucleic acid or amino acid sequence is at least 75% of a control sequence as measured by one of the known programs described herein (eg, BLAST) using standard parameters. A polynucleotide or amino acid sequence that may comprise a sequence having 80% or 90% sequence identity. The sequence identity is preferably at least 95%, more preferably at least 98%, and most preferably at least 99%. In some embodiments, the sequence of interest is approximately the same length as compared to the control sequence, ie, approximately the same number of consecutive amino acid residues (for polypeptide sequences) or nucleotide bases (for polynucleotide sequences). Consists of.

  Sequence identity can be readily determined by various methods known in the art. For example, the BLASTN program (for nucleotide sequences) uses, as a default, word length (W) 11, expectation (E) 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, as a default, word length (W) 3, expectation (E) 10, and BLOSUM62 scoring matrix (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)). The percent sequence identity is determined by comparing two optimally aligned sequences in a comparison window, where the polynucleotide sequence portion in the comparison window is for two optimally aligned sequences. It may contain additions or deletions compared to a control sequence (which does not contain additions or deletions). The percentage is calculated by determining the number of positions where the same nucleobase or amino acid residue is present in both sequences to obtain the number of matching positions, and the total number of matching positions in the window is compared. Dividing by the number of positions and multiplying it by 100 gives the percent sequence identity.

  A cell has been “transformed” or “transfected” by an exogenous or heterologous polynucleotide when such a polynucleotide has been introduced into the cell. The transforming polynucleotide may or may not be integrated (covalently) into the genome of the cell. In prokaryotes, such as yeast and mammalian cells, the transforming polynucleotide can be maintained on an episomal component such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming polynucleotide is integrated into the chromosome so that it is inherited by daughter cells via chromosome replication. This stability is evidenced by the ability of eukaryotic cells to establish cell lines or clones that have a population of daughter cells that contain the transforming polynucleotide. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that can be stably propagated in vitro for many generations.

  The term “vector” or “construct” means sequence elements arranged in a pattern so that expression of a gene / gene product operably linked to a polynucleotide sequence component can be predictably controlled. To do. Typically, they are heritable polynucleotide sequences in a portion of an external polynucleotide sequence that can be inserted to introduce external DNA into a host cell and promote its replication and / or transcription (eg, , Plasmid or virus).

  Cloning vectors are polynucleotide sequences (typically plasmids or phages) that are autonomously replicable in a host cell and are characterized by recognition sites for one or a few restriction endonucleases. External polynucleotide sequence fragments can be inserted into the vector at these sites for fragment replication and cloning. The vector can include one or more markers suitable for use in identifying transformed cells. For example, the marker can provide tetracycline or ampicillin resistance.

  Expression vectors are similar to cloning vectors, but are capable of inducing expression of the polynucleotide sequence cloned therein after transformation into the host. The cloned polynucleotide sequence is usually under the control of (ie, operably linked to) any control sequence such as a promoter or enhancer. The promoter sequence can be constitutive, inducible or repressible.

III. Ribosomal polynucleotides for expressing mammalian 18S rRNA The present invention provides synthetic (isolated or modified) ribosomal polynucleotides or nucleic acid molecules for studying mammalian 18S rRNA expression and associated ribosomal activity. The ribosomal polynucleotides described herein include the rDNA and rRNA nucleotide sequences described herein, and combinations or substantially identical variants thereof. In some embodiments, the polynucleotide encodes (for a DNA sequence) or comprises a full-length or partial mammalian (eg, mouse or human) 18S rRNA nucleotide sequence, or a substantially identical variant thereof. (For RNA sequences). In certain embodiments, the polynucleotide encodes a full-length or partial SEQ ID NO: 23, or a substantially identical variant thereof, having one or more specific substitutions described herein, Or include. In some of these embodiments, the synthetic polynucleotide comprises a fragment of full length 18S rDNA or rRNA sequence and may have one or more specific substitutions. Thus, the ribosomal polynucleotides described herein are mammalian (eg, mouse or human) rRNA nucleotide sequences, rDNA nucleotide sequences, or pre-rRNA nucleotide sequences, fragments thereof, or substantially identical mutations thereof. May contain body.

  In one aspect, an isolated or synthetic ribosomal polynucleotide that includes or encodes a pactamycin resistant 18S rRNA or fragment thereof is provided. In some preferred embodiments, the encoded pactamycin resistant 18S rRNA is a mammalian 18S rRNA. Pactamycin resistance is typically provided by one or more single base substitutions at several specific conserved bases at the E site of the encoded 18S rRNA. Using the mouse 18S rDNA sequence (SEQ ID NO: 23) (Accession No. JQ247698) as a control sequence, these bases include G963, A964, C1065 and C1066. Some embodiments described herein relate to ribosomal polynucleotides or “synthetic polynucleotides” that comprise or encode 18S rRNA of pactamicin resistant mammals. These polynucleotides typically correspond to positions G963, A964, C1065 and C1066 in the mouse 18S rDNA sequence (SEQ ID NO: 23), a sequence substantially identical thereto, or a sequence containing substitutions or mutations thereof. Contains a mammalian 18S rDNA sequence with one or more mutations in position.

  As noted above, the bases for substitution are conserved among different mammalian species, and the exact position of these bases in other mammalian species can be readily determined (eg, by sequence alignment). . Thus, as used herein, the nucleotide bases or positions corresponding to G963, A964, C1065, and C1066 in the mouse 18S rDNA sequence (SEQ ID NO: 23) are those that encode a mammalian 18S rRNA or complementary sequence. Of these conserved bases present in the polynucleotide sequence. For example, the corresponding bases in another mouse 18S rDNA sequence, accession number X00686 (SEQ ID NO: 34; Raynal et al., FEBS Lett. 167: 263-268, 1984) are G962, A963, C1064 and C1065, respectively. . Similarly, the corresponding bases of the human 18S rDNA sequence (SEQ ID NO: 24) (McCallum et al. Biochem. J. 1985; 232: 725-733, 1985; accession number X03205) are G961, A962, C1063 and C1064, respectively. It is. The exact location of these conserved bases in various other mammalian 18S rRNA or rDNA sequences can also be readily determined. These include, for example, mouse 18S rDNA sequence (Accession No. NR — 003278; SEQ ID NO: 35), rat 18S rDNA sequence (Accession No. X01117, SEQ ID NO: 36; and M11188, SEQ ID NO: 37), rabbit 18S rDNA sequence ( Accession No. X06778, SEQ ID NO: 38), and human 18S rDNA sequence (Accession No. K03432, SEQ ID NO: 39; and M10098, SEQ ID NO: 40).

  In certain embodiments, a ribosomal polynucleotide described herein comprises a full-length mammalian 18S rRNA having one or more specific substitutions that provide pactamycin resistance as described herein, or a coding Can be In certain other embodiments, the synthetic polynucleotide may comprise or encode 50 or fewer contiguous nucleotides of a mammalian 18S rRNA containing one or more specific substitutions that provide pactamycin resistance. In certain other embodiments, the polynucleotide comprises or encodes 100 or fewer contiguous nucleotides of mammalian 18S rRNA containing one or more specific substitutions. In yet other embodiments, the polynucleotide comprises 250, 500, 750, 1000, 1500 or more contiguous nucleotides of mammalian 18S rRNA containing one or more of the specific substitutions described above that provide pactamicin resistance. Contains or encodes. In any of these embodiments, the synthetic polynucleotide can also include a substantially identical or complementary sequence of the full length 18S rRNA sequence or fragment sequence described herein.

  In certain other embodiments, the polynucleotide is at least 80%, 90%, 95%, or 99% identical to SEQ ID NO: 23, and is 1 at a position corresponding to positions G963, A964, C1065, and C1066 of SEQ ID NO: 23. It contains or is complementary to a sequence containing or more substitutions. In some of these embodiments, the mutation is at a position corresponding to position G963 of SEQ ID NO: 23. For example, the polynucleotide can comprise or encode an 18S rRNA sequence having a G → A substitution at a position corresponding to G963 of SEQ ID NO: 23. In certain embodiments, the polynucleotide comprises a sequence that is identical or complementary to a mouse 18S rDNA or human 18S rDNA sequence that does not contain one or more specific substitutions described herein. As noted above, the synthetic polynucleotides described herein also include fragments of these sequences having one or more specific substitutions.

  The ribosomal polynucleotides described herein can be single-stranded, double-stranded, triple-stranded (triplex), linear or circular. They may comprise one or more nucleotide derivatives or analogs as described above (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more analogs or derivative nucleotides). . In some embodiments, the polynucleotide consists entirely of one or more analogs or derivative nucleotides, and sometimes the polynucleotide is about 50% or less, about 25% or less, about 10% or less. Or about 5% or less of an analog or derivative nucleotide base. One or more nucleotides in the analog or derivative nucleic acid are nucleobase or backbone modifications such as ribose or phosphate modifications (eg, ribose peptide nucleic acid (PNA) or phosphorothioate linkages) compared to RNA or DNA nucleotides. Can be included. Nucleotide analogs and derivatives are known to those skilled in the art, and non-limiting examples of such modifications are US Pat. Nos. 4,469,863; 5,536,821; No. 306; No. 5,637,683; No. 5,637,684; No. 5,700,922; No. 5,717,083; No. 5,719,262; No. 5,739,308; No. 5,773,601; No. 5,886,165; No. 5,929,226; No. 5,977,296; No. 6,140,482 No .; 6,455,308; and WIPO Publication Nos. WO00 / 56746 and WO01 / 14398. Methods for the synthesis of nucleic acids containing such analogs or derivatives are also described, for example, in the above cited patent documents and US Pat. Nos. 6,455,308; 5,614,622; 5,739, No. 314; No. 5,955,599; No. 5,962,674; No. 6,117,992; and WO 00/75372.

IV. Vectors for expressing pactamycin resistant 18S rRNA A polynucleotide encoding a mammalian 18S rRNA described herein can be inserted into an expression vector for introduction into a mammalian host cell. These vectors are typically circular and may contain selectable markers, origins of replication, and other elements in addition to the polynucleotide encoding 18S rRNA. For example, the vector can include a selectable marker. A selectable marker allows the selection of cells into which the vector has been introduced and / or deliberately integrated. In certain embodiments, the selectable marker can be a polynucleotide encoding a protein or enzyme that provides a visually distinguishable characteristic to the cell. For example, as exemplified herein, a vector may have a selectable marker that encodes a Renilla luciferase reporter enzyme. Other examples include jellyfish green fluorescent protein (GFP) and bacterial β-galactosidase. In some other embodiments, the selectable marker for identifying host cells into which the vector has been introduced and / or stably harbored can be an antibiotic resistance gene. Examples of such markers include antibiotic resistance genes for neomycin, chloramphenicol, blasticidin, hygromycin, and zeocin.

  In addition to sequences encoding pactamycin resistant 18S rRNA, the vector may also have other rDNA sequences that may be required for proper rRNA transcription and processing, as well as proper ribosome accumulation and function. For example, certain vectors described herein may further have sequences corresponding to the 5'-ETS and ITS elements of the precursor rRNA sequence. Specific vectors for expressing pactamicin resistant mammalian 18S rRNA are exemplified in the following examples (eg, Example 4).

  Another component of the expression vector is the origin of replication. An origin of replication is a unique DNA segment that contains multiple short repeats that are recognized by multimeric proteins that bind to the origin and play an important role in assembling DNA replication enzymes at the origin site. Suitable origins of replication for private use in the vectors described herein include, for example, EBV oriP, SV40, E. coli oriC, colE1 plasmid origin, ARS, and the like. Another useful element in expression vectors is multiple cloning sites or polylinkers. Synthetic DNA encoding a series of restriction endonuclease recognition sites is inserted into a plasmid vector, eg, downstream of a promoter element. These sites are designed to conveniently clone the DNA at a specific location in the vector.

A polynucleotide or vector for expressing the modified 18S rRNA described herein can be easily constructed according to a method known in the field of molecular biology in view of the description herein. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (3 ^rd ed., 2001); Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); and Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss, Inc. (4 ^th ed., 2000). In general, expression vectors are constructed by inserting a polynucleotide encoding a pactamycin resistant 18S rRNA, a sequence encoding a selectable marker, and any other elements described herein into a suitable vector backbone. The In order to produce the vector, the polynucleotide described above can be inserted into various known plasmids for transforming mammalian host cells. Such known plasmids include, for example, pRL-CMV (Promega), BPV, EBV, vaccinia virus based vectors, SV40, 2-micron circle, pcDNA3.1, pcDNA3.1 / GS, pYES2 / GS, pMT, pIND , PIND (Sp1), pVgRXR (Invitrogen), etc., or derivatives thereof. All of these plasmids have been described and are known in the art (Botstein et al., Miami Wntr. SyTnp. 19: 265-274, 1982; Broach, In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell 28: 203-204, 1982; Dilon et at., J. Clin. Hematol. Oncol. 10: 39-48, 1980; and Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980).

V. Host cells expressing pactamycin resistant 18S rRNA Provided are constructed mammalian cells that express pactamycin resistant 18S rRNA. By using the polynucleotide molecule or expression vector described above, various mammalian cells introduce the expression vector described herein or stably integrate the rDNA described herein into the host genome. Can be used. A polynucleotide encoding pactamycin resistant 18S rRNA or the above expression vector is introduced into an appropriate host cell (eg, a mammalian cell such as a mouse N2a cell or a CHO cell) by any means known in the art. Can be done. The cell may transiently or stably express pactamycin resistant 18S rRNA.

  Preferably, the host cell for expressing a mammalian 18S rRNA described herein is a eukaryotic cell, eg, a mammalian cell. A eukaryotic vector / host system, preferably a mammalian expression system, allows for suitable post-translational modifications of the expressed mammalian protein, eg, suitable processing, glycosylation, phosphorylation, and advantageous of the primary transcript. In some cases, secretion of the expression product occurs. Thus, eukaryotic cells such as mammalian cells are preferred host cells for introducing the polynucleotides or expression vectors described above. One specific example is the mouse neuro 2a (N2a) cell detailed in the examples below. Other suitable cells include animal cells (eg, insects, rodents, cattle, goats, rabbits, sheep, non-human primates, cells derived from humans) and plant cells (eg, rice, corn, cotton , Tobacco, tomatoes, potatoes, etc.). Other specific examples of such host cell lines include CHO, BHK, HEK293, VERO, HeLa, COS, MDCK, and W138.

  Host cells for expressing the synthetic 18S rRNA described herein other than mammalian cells may be yeast cells or plant cells. Yeast or plant cells suitable for stable integration and expression of the transgene can be used for these applications by introducing a polynucleotide encoding 18S rRNA into the host via a yeast or plant expression vector. Examples of suitable insect cells include cells from Drosophila larvae. When using insect cells, the polynucleotide can be introduced into the cells by a suitable expression vector. For example, baculovirus vectors may be those previously reported in the art (Jasny, Science 238: 1653, 1987; and Miller et al., In: Genetic Engineering (1986), Setlow, JK, et al., Eds , Plenum, Vol. 8, pp. 277-297). When insect cells are used as hosts, the Drosophila-alcohol dehydrogenase promoter can optionally be used in expression vectors for introducing polynucleotides (Rubin, Science 240: 1453-1459, 1988).

  Any convenient protocol can be used to introduce a vector expressing pactamycin resistant 18S rRNA into a host cell in vitro or in vivo, depending on the location of the host cell. In certain embodiments, the expression vector is transiently transfected into a host cell (eg, a cultured cell line such as N2a), as illustrated in the Examples below. This can be accomplished using conventional methods, for example, by the following protocol exemplified herein. In certain other embodiments, the expression vector can be stably integrated into the host genome. In general, a polynucleotide can be transfected into a host cell after appropriate restriction enzyme digestion to produce a free end homologous to the host chromosome. Expression vectors can be introduced into host cells by standard protocols commonly used in the art. For example, the vector can be transfected into the host cell by conventional mechanical techniques such as calcium phosphate coprecipitation, microinjection or electroporation, insertion of a plasmid in liposomes, and viral vectors. All of these techniques are known and commonly used in the art. For example, Freshney ,; Sambrook et al.,; And Brent et al., Supra). Host cells with the transfected recombinant expression vector can be identified and isolated using a selectable marker present on the vector. Many recipient cells can be grown in a medium that selects for cells containing the vector.

  In certain embodiments, when the host cell is an isolated cell, the expression vector is introduced directly into the cell, for example using standard transformation techniques, under cell culture conditions that permit host cell viability. Can be done. Such techniques include, but are not necessarily limited to, viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun method, calcium phosphate precipitation, direct microinjection, viral vector delivery and the like. The choice of method generally depends on the cell type to be transformed and the conditions under which the transformation occurs (ie, in vitro, ex vivo, or in vivo). A general discussion of these methods can be found, for example, in Brent et al above.

  Alternatively, when the host cell or cell is part of a multicellular organism, the target vector can be administered to the organism or host in such a way that the target vector can enter the host cell, for example, by in vivo or ex vivo protocols. . “In vivo” means that the target construct is administered into the animal body. “Ex vivo” means that a cell or tissue is modified outside the body. Such cells or tissues are generally returned to the living body. Methods of administering nucleic acid constructs are known in the art. For example, nucleic acid constructs can be obtained using viral vectors (Monahan et al., Gene Therapy 4: 40-49, 1997; Onodera et al., Blood 91: 30-36, 1998), “naked DNA” Can be delivered with cationic lipids (Goddard, et al, Gene Therapy, 4: 1231-1236, 1997; Gorman et al., Gene Therapy 4: 983-992, 1997; Chadwick et al., Gene Therapy 4: 937-942, 1997; Gokhale et al., Gene Therapy 4: 1289-1299, 1997; Gao and Huang, Gene Therapy 2: 710-722, 1995). Techniques well known in the art for transfection of cells (see above) can be used for ex vivo administration of nucleic acid constructs. The exact formulation, route of administration and dosage can be chosen empirically. See, for example, Fingl et al., 1975, in The Pharmacological Basis of Therapeutics, Ch. 1 p 1.

VI. Modification of rRNA for Enhanced Translational Efficiency or Altered Ribosome Function The polynucleotides or related expression vectors encoding synthetic mammalian 18S rRNA described herein may have improved or altered ribosome function and Useful for identifying mutations that result in translational activity (eg, in 18S rDNA sequences). The 18S rRNA expression system described herein can also be used for functional analysis of naturally occurring variants in 18S rRNA. They are also suitable for modifying rRNA sequences to produce ribosomes with improved properties, such as enhanced translation efficiency. For example, to enhance the expression of a particular polypeptide of interest, a particular mutation is introduced into the 18S rRNA for enhanced base pair interaction between the rRNA encoding the polypeptide of interest and the mRNA. Can be done. By stopping endogenous 18S rRNA function, the pactamycin resistant 18S rRNA expression system described herein allows screening and selecting for mutations in 18S rRNA that can increase expression of the protein of interest. Similarly, rRNA sequences, particularly 18S rRNA, are used for modified sequences that result in ribosomes having altered accumulation or enhanced translational activity (eg, interaction with tRNA) that are not limited to any particular mRNA. Modifications and selections can be made.

  Thus, methods are provided to study the functional consequences of mutations or naturally occurring mutations in 18S rRNA sequences. In certain embodiments, the effects of naturally occurring mutations in the 18S rRNA sequence are tested by use of the expression system described herein. Typically, a polynucleotide sequence having a naturally occurring variant (eg, a mouse or human variant 18S rRNA sequence described herein) will introduce pactamicin resistance as described herein, eg, the sequence Modified to introduce a substitution corresponding to G963A at number 23. The modified polynucleotide present in the expression vector is then introduced into the host cell for functional analysis of rRNA processing and ribosome function in the presence of pactamycin. Any difference in the activity tested compared to the activity of the wild-type or control 18S rRNA sequence (eg, SEQ ID NO: 23) is between sequence variation and the observed change in rRNA processing and / or ribosome activity. The structure-function correlation can be shown.

  In some other embodiments, the 18S rRNA expression system is used to screen for mutations in the 18S rRNA sequence that result in altered ribosome function and / or translational activity. In these embodiments, the polynucleotide sequence encoding the 18S rRNA sequence of a pactamycin resistant mammal or fragment thereof is further modified to generate a candidate 18S rDNA sequence by random or site-directed mutagenesis. When introduced into a host cell, the candidate or mutant 18S rDNA sequence is screened and selected to identify mutations that provide enhanced translation efficiency or altered ribosome function. Mutagenesis and subsequent selection can be performed using standard molecular biology techniques described herein.

  Still other aspects described herein are directed to modifying 18S rRNA sequences for enhanced expression of a particular polypeptide of interest. In these embodiments, random or site-specific mutations are introduced into the polynucleotide sequence encoding pactamycin resistant 18S rRNA if the mutation does not affect the pactamycin resistant activity of the encoded 18S rRNA. . Random mutations in the sequence encoding rRNA can be generated using methods known in the art. For example, DNA shuffling as described by Stemmer (Nature 370: 389-391, 1994) can be readily used to introduce random mutations into rDNA sequences. Alternatively, error prone amplification can be used to introduce random mutations as described, for example, in Bartell and Szostak, Science 261: 1411, 1993. Additional techniques for generating random mutations can also be used. For example, sequences encoding 18S rRNA can also be found in cassette mutagenesis (Hutchison et al., Methods Enzymol. 202: 356-390, 1991), recursive ensemble mutagenesis (Arkin et al. , Proc. Natl. Acad. Sci. USA 89: 7811-7815, 1992), exponential ensemble mutagenesis (Delegrave et al., Biotechnol. Res. 11: 1548-1552, 1993), And can be mutagenized by sexual PCR mutagenesis (Stemmer et al., Proc. Natl. Acad. Sci. USA 91: 10747-10751, 1994).

  Candidate 18S rDNA sequence variants other than random mutagenesis that are selected and screened for enhanced translational activity or altered ribosome function can also be generated by targeting or site-directed mutagenesis. Site-directed mutagenesis in 18S rDNA sequences can be performed by methods known in the art, for example, the method described in Arnold Curr. Opinion Biotechnol 4: 450-455, 1993. In certain embodiments, 18S rDNA sequence variants are generated using site-directed mutagenesis using oligonucleotide-directed mutagenesis. Oligonucleotide mutagenesis is described, for example, in Reidhaar-Olson, Science 241: 53-57, 1988. Briefly, a number of double stranded oligonucleotides having one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized. Clones containing the mutated DNA are recovered and then the activity of the rRNA that they encode is evaluated. Another method for generating mutant 18S rDNA sequences is assembly PCR. Assembly PCR involves the assembly of PCR products from a mixture of small pieces of DNA. A number of different PCR reactions occur in parallel in the same vial, with the product of one reaction yielding the product of another reaction. Assembly PCR is described, for example, in US Pat. No. 5,965,408.

  For screening and selection of mutations having the desired phenotype, a mutant 18S rDNA sequence encoding a pactamycin resistant 18S rRNA and further having a site-specific mutation or a random mutation is obtained via the expression vector described herein. Introduced into the host cell. The cells are then cultured in the presence of pactamycin to assess the level of expression of the polypeptide of interest or other translational activity of accumulated ribosomes. In order to identify modified 18S rRNA for enhanced expression of a specific polypeptide, the expression level of the specific polypeptide in the cell is tested to express pactamycin resistant 18S rRNA, but include other mutations in the 18S rRNA Compare the expression level of the same polypeptide in no control cells. A mutation in the 18S rRNA that results in an enhanced level of translation of the polypeptide of interest indicates that the host cell has produced ribosomes with enhanced expression of the polypeptide of interest, as compared to those without the mutation. . As described in detail below, the methods described herein are suitable for enhancing the expression level of various polypeptides or proteins of interest. The polypeptide of interest can be either endogenous or exogenous to the host cell.

  In addition to selecting modified 18S rRNA sequences that provide enhanced expression levels of the polypeptide of interest, the 18S rRNA expression system described herein can also be used to generate ribosomes with other altered functions. Can be used. In these embodiments, a polynucleotide encoding a pactamycin resistant 18S rRNA and having the site-specific mutation or random mutation described above can be tested in a host cell for any altered ribosome activity resulting from the mutation. Ribosome activities that can be monitored include, for example, ribosome accumulation, interaction with tRNA and / or mRNA, translation initiation, and elongation and termination. Various ribosome functions can be monitored using techniques or assays routinely used in the art. For example, rRNA maturation and ribosome accumulation in mammalian cells is described, for example, by Pestov et al., Curr. Protoc. Cell Biol. 2008, Chapter 22: Unit 22.11; Champney, Methods Mol. Med. 142: 63-73, 2008 And can be tested using the techniques described in Klostermeier et al., Nucleic Acids Res. 32: 2707-15, 2004. Ribosome binding to mRNA can be tested using an assay similar to that described in, for example, Day et al., J. Bacteriol. 186: 6864-6875, 2004. Ribosome binding to the ER membrane during protein transfer is described, for example, by Prinz et al., EMBO J. 19: 1900-1906, 2000; and Kalies et al., J. Cell Biol. 126: 925-934, 1994. Can be monitored using the assay described in. The binding of aminoacyl-tRNA to ribosomes can be tested according to standard binding assays well known in the art, e.g., Agafonov et al., EMBO Rep. 2: 399-402, 2001; Ashraf et al., RNA. 5: 188-194, 1999, and Lill et al., Methods Enzymol. 164: 597-611, 1988. Possible effects of 18S rRNA mutations on other aspects of protein synthesis (eg, translation initiation, extension, termination and peptide release) can also be monitored using routine assays. For example, Burakovsky et al., RNA. 16: 1848-53, 2010; Sternberg et al., Nat. Struct. Mol. Biol. 16: 861-8, 2009; Van Dyke et al., Nucleic Acids Res. 37: 6116-25, 2009; Sunohara et al., J. Biol Chem. 279: 15368-75, 2004; Khan et al., Biochim. Biophys. Acta. 1779: 622-7, 2008; Anderson et al., J. Biol. Chem. 282: 14752-60, 2007; Merill et al., J. Virol. 80: 6936-42, 2006; Brunelle et al., RNA. 14: 1526-31, 2008; Rawat et al., J Mol. Biol. 357: 1144-53, 2006; and Gong et al., J Bacteriol. 189: 3147-55, 2007.

VII. Polypeptide or protein expression vectors for enhanced translation and host cells that express pactamycin resistant 18S rRNA are useful for enhancing the level of expression of an important polypeptide or protein of interest. The protein of interest can be a polypeptide having medical or industrial application. In certain embodiments, the polypeptide or protein of interest is one that encodes a therapeutic protein. Examples of therapeutic proteins include Factor VIII, Factor IX, β-globin, low density lipoprotein receptor, adenosine deaminase, purine nucleoside phosphorylase, sphingomyelinase, glucocerebrosidase, cystic fibrosis transmembrane conductance regulator, α-antitrypsin, CD-18, ornithine transcarbamylase, argininosuccinate synthetase, phenylalanine hydroxylase, branched α-keto acid dehydrogenase, fumaryl acetoacetate hydrolase, glucose 6-phosphatase, α-L-fucosidase, β- Examples include glucuronidase, α-L-iduronidase, galactose 1-phosphate uridyltransferase, interleukin, cytokine, small molecule peptide and the like. Other therapeutic proteins that can be expressed from the polynucleotide of interest inserted in the engineered host cells described herein include, for example, Herceptin®, bacteria or viruses (eg, E. coli, Pseudomonas aeruginosa, yellow Polypeptide antigens from various pathogens such as diseases caused by staphylococci, malaria, HIV, rabies virus, HBV, and cytomegalovirus) and other proteins such as lactoferrin, thioredoxin and beta-casein vaccines included.

  Further examples of proteins of interest include insulin, erythropoietin, tissue plasminogen activator (tPA), urokinase, streptokinase, neutropoiesis stimulating protein (filgastim or granulocyte colony stimulating factor (G-CSF) ), Thrombopoietin (TPO), growth hormone, hemoglobin, insulin secretory factor, imiglucerase, salgramostim, endothelial factor, soluble CD4, and antibodies and / or antigen-binding fragments thereof (eg, FAb) (eg, orthoclones) OKT-e (anti-CD3), GPIIb / IIa monoclonal antibody), ciliary neurotrophic factor (CNTF), granulocyte macrophage colony stimulating factor (GM-CSF), brain-derived neurotrophic factor (BDNF), parathyroid Gland hormone (PTH) -like hormone, insulinotropic hormone, insulin-like growth factor-1 (IGF-1), platelet derived growth factor (PDGF), epidermal growth factor (EGF), acidic fibroblast growth factor, Basic fibroblast growth factor, transforming growth factor β, nerve growth factor (NGF), interferon (IFN) (eg, IFN-α2b, IFN-α2a, IFN-αN1, IFN-β1b, IFN-γ), Interleukins (eg IL-1, IL-2, IL-8), tumor necrosis factor (TNF) (eg TNF-α, TNF-β), transforming growth factors-α and -β, catalase, calcitonin, Arginase, phenylalanine ammonia lyase, L-asparaginase, pepsin, uricase, tryp , Chymotrypsin, elastase, carboxypeptidase, lactase, sucrase, endogenous factor, vasoactive intestinal peptide (VIP), calcitonin, Ob gene product, cholecystokinin (CCK), serotonin, and glucagon. There is no need to be limited.

  Suitable polypeptides of interest also include certain membrane proteins or other intracellular proteins. Examples of membrane proteins include, but need not be limited to, adrenergic receptor, serotonin receptor, low density lipoprotein receptor, CD-18, sarcoglycan (deleted in muscular dystrophy), and the like. Absent. Useful intracellular proteins include proteins that are primarily localized within the intracellular compartment or that exhibit the desired biological activity within the cell. Such intracellular proteins can include fumaryl acetoacetate hydrolase (FAH) that is missing in subjects with genetic tyrosinemia type 1. Other specific examples of intracellular proteins include antiviral proteins (eg, proteins that can provide inhibition of viral replication or selective cell death of infected cells), collagen, ie recessive dystrophic epidermal blisters Structural proteins such as the type VII collagen COL7A1 gene that are deleted in dystrophy (RDEB), as well as dystrophin that is deleted in muscular dystrophy.

Examples The following examples are provided for further illustration, but are not intended to limit the scope of the invention. Other variations will be readily apparent to those skilled in the art and are encompassed by the appended claims.

Example 1 Identification of Large 18S rRNA Variants Mammalian cells contain hundreds of non-identical rDNA genes. These sequence variants provide ribosome diversity and may have a functional impact. Some sequences were cloned from mouse N2a genomic DNA. rDNA fragments, which are located about 700 nucleotides upstream and 70 nucleotides downstream of 18S rDNA, respectively. 1 and rDNA. PCR amplification was performed using 2 (Table 1) as a primer. Six independent clones were sequenced and found to be> 99% identity to each other. However, none of these sequences are completely identical to each other or to two different 18S rDNA sequences in the NCBI nucleotide database (X82564, NR_003278, X00686; sequences X82564 and NR_003278 contain the same 18S sequence). In addition to some unique variants that differ from the published sequence, the sequence identified in this experiment contains one nucleotide difference in helix H41a and two single nucleotide insertions (one is an extension segment ( (Expansion segment) 3) and the other (in helix H30).

  For the expression system, the 18S rDNA gene sequence containing the most common sequence variation at each nucleotide position was used to provide a control for future studies of rRNA sequences containing fewer common sequence variations. In order to evaluate the cloning sequences against a population of 18S rDNA sequences, they were compared to the resulting sequences using the collected genomic PCR products as templates. Except for a significant bias in the PCR reaction, the sequence of genomic sequences collected should represent the major genomic variation at each nucleotide. Two independent PCR reactions (PCR1 and PCR2) were performed and the products were directly sequenced. Comparison of the collected genomic sequence with that of the clones isolated in this experiment revealed that one of the clones contained the same sequence as the collected genomic sequence. This clone (Accession number JQ247698; SEQ ID NO: 23) was selected for subsequent experiments. It was 1,871 nucleotides long and contained the three common mutations described above, but did not contain other mutations from the published sequence X00686.1 (SEQ ID NO: 34).

Example 2 Construction of 18S rRNA Expression Construct An 18S rRNA expression system was developed to experiment with the biosynthesis of the 40S ribosomal subunit and the role of this RNA in the translation initiation process. It was anticipated that the mouse rDNA genomic fragment might require proper processing at positions A ′, A ₀ , 1, and 2 (FIG. 1b). The 18S rDNA clones identified above are used to create expression constructs containing 5′ETS, 18S rDNA, and ITS1, which contain all of these positions (FIG. 2a). Constructs were made using the pol-I promoter and 3′ETS, or the cytomegalovirus (CMV) promoter and SV40 poly (A) signal. A construct having a deletion at the 5 ′ end of 5′ETS and the 3 ′ end of ITS1, ie, having a less authentic spacer sequence adjacent to the processed (mature) end of 18S rRNA. Were also tested. These various constructs were transfected into N2a cells and the expression of synthetic 18S rRNA was determined by Northern blot using an oligonucleotide probe against a 24 nt hybridization tag cloned into the extended segment 3 of 18S rDNA. . The results show that only constructs with full-length 5 ′ ETS containing positions A ′ and A ₀ were verified by comparing size with 18S rRNA transcripts in vitro, appropriately processed 18S rRNA (p18S.1). And 18S.2 (Pol-1 and CMV); FIG. In contrast, ITS1, which contains 3 ′ processing sites 2b and 2c, is mostly composed of p18S. Even if deleted in 2 (Pol-1 and CMV), this construct appears to be unnecessary because it produces a band corresponding to mature 18S rRNA.

  Expression of mature 18S rRNA was observed to occur with both pol-I and CMV promoters. However, its expression level was higher when transcription was mediated by pol-I (FIG. 2b). The results indicate that the processing site in 5'ETS is sufficient for 18S rRNA processing and that processing does not require transcription to occur by RNA polymerase I. Due to the low yield observed in pol-II transcription of 18S rRNA, the pol-I vector was exclusively used for all subsequent experiments.

  Construct p18S. 1 and p18S. Correct processing of synthetic 18S rRNA precursor transcripts from 2 was demonstrated by measuring blots with α-tag probes to identify these 18S rRNAs. The same blot was reprobed with a low specific activity oligonucleotide probe (α-18S) that recognizes both synthetic and endogenous 18S rRNA (FIGS. 7a, b, c). The results show that mature synthetic and endogenous 18S bands can be superimposed. To demonstrate the removal of the spacer region from the processed RNA, Northern blotting was performed with 5′ETS (α-5′ETS) and ITS1 (5 ′ and 3 ′ immediately after sites 1 and 2, respectively, ITS1 ( A probe complementary to the sequence in α-ITS1) was used (FIG. 7d, e). Both blots were undetectable by hybridization with the mature tagged transcript, but hybridized with a smaller amount of the precursor transcript, showing results consistent with the removal of the spacer region from the mature 18S rRNA. It was. For constructs with deletions in the spacer region, the size of the unprocessed and partially processed transcript is consistent with the expected size when compared to the pre-rRNA species.

  To monitor the relative levels of synthetic 18S rRNA after transfection, a time course experiment was performed. For these experiments, cells were transfected with a full length pol-I construct (p18S.1) that produced a processed 18S rRNA, and 18S rRNA expression was expressed at various time points up to 72 hours post-transfection. Collected and monitored, then probed for hybridization tags (FIG. 2c). For this construct, maximum expression of synthetic 18S rRNA was observed as a percentage of total 18S rRNA 36-48 hours after transfection. All subsequent experiments were performed 48 hours after transfection unless otherwise noted.

  To determine whether the processed synthetic 18S rRNA binds to the ribosome, Northern blot analysis was performed using p18S. 1 was performed on whole cell lysates of transfected cells and cell lysate pellet (P) and supernatant (S) fractions (FIGS. 8a, b). As described above, unprocessed and processed synthetic 18S rRNA was present in total RNA prepared from total cell lysates (FIGS. 8a, b; right). However, in the fractionated material, only processed rRNA was found in P100 pellets containing precipitated ribosomes. No processed RNA was found in the supernatant fraction containing lower density cytoplasmic material. These fractionated RNA samples were also compared to synthetic in vitro transcribed rRNA by probing with both α-tag and α-18S probes to determine relative abundance. Synthetic 18S rRNA in cells 48 hours after transfection was estimated to be about 10-15% of total 18S rRNA. p18S. Fractionation of lysates from cells transfected with 2 (pol-I) gave similar results (FIG. 8c), with synthetic 18S rRNA lacking ITS1 being incorporated into the ribosomal subunit. Was suggested. In summary, these experiments were performed using p18S. 1 and p18S. 2 shows that the synthetic 18S rRNA from 2 was correctly processed and incorporated into the 40S subunit. They also indicate that ITS1 is unnecessary for the formation of mature 18S rRNA.

Region 5 '5'ETS including and _{A 0} processing site' Analysis A of Example 3 5'ETS sequence, it is clear that it is necessary in the processing of 18S rRNA (Fig. 1b); however, mammalian rDNA Little is known about the importance of sequences located 3 'to these sites of the gene. The length of these 3 'sequences in mammalian genes differs from other eukaryotic rDNA genes that are substantially shorter. In order to investigate the potential contribution of these additional sequences in mammals, several internal deletions were made, including deletions individually and in combination to remove positions A ′ and A ₀ ( FIG. 3a).

Northern blots of RNA extracted from cells transfected with an internal deletion construct were hybridized using oligonucleotide probes to the hybridization tag. The results demonstrated that deletions other than cleavage positions A ′ or A ₀ prevented processing (p18S.9, S.10, and S.11; FIG. 3b). However, deletion of the extended 3 ′ region of 5′ETS had little effect on 18S rRNA maturation (p18S.7). This result suggests that this region lacks an important cleavage site and other points of interaction with the rRNA processing factor.

Cleavage at positions A ′, A ₀ , and 1 requires U3 snoRNA, and in mice the putative binding site for U3 snoRNA is in two regions in the snoRNA called the 3 ′ and 5 ′ hinge regions An assumption was made based on matching complementary sequences. Based on these predictions and observations, p18S. 9, S.M. 10 and S.E. 11 deletion of several possible binding sites for the U3 snoRNA 3 ′ hinge at nucleotides 667-673, 1032-1038 and 1117-1123, and p18S. 8, p18S. 9 and p18S. The deletion at 10 eliminated a potential binding site for the 5 'hinge at nucleotides 1552-1560. In all these constructs, the deletion prevented processing. p18S. Deletion in 8 'excluding 9-nt putative binding sites for the hinge, cutting position A' 5 of U3 snoRNA both and _{A 0} comprises. To specifically test the need for this putative binding site for processing, construct p18S. 8Δ and p18S. The 9-nt sequence in 5′ETS at each 8 m was deleted or mutated (FIG. 3c). Northern blot analysis of RNA from cells transfected with these constructs indicates that disruption of the 9-nt sequence in both constructs almost completely prevented processing. This result supports this 9-nt sequence binding to the U3 snoRNA 5 ′ hinge region.

Since the A ′ cleavage is dependent on U3 snoRNA, the construct p18S. 8 and p18S. It is noted that removal of the putative 5 ′ hinge region in 9 results in multiple immature rRNA bands indicating incomplete cleavage at A ′ (FIG. 3b). It is clear that some A ′ cleavages can occur without producing a sequence complementary to the U3 snoRNA 5 ′ hinge. In contrast, p18S. Lacks position A ′ and a potential 3 ′ hinge binding site. 11, and p18S. Lacking binding sites for both hinge regions. 10 results in a primary transcript that are not cut, A ', 3' when the hinge region, or a combination of these sites, there is no, indicating that there is no discernible cleavage at A _0.

Example 4 Identification of Pactamycin Resistance Mutation and Confirmation of Subunit Function The ability to selectively monitor in vivo translation from subunits containing synthetic 18S rRNA is useful for distinguishing modifications and activities of endogenous ribosomal subunits. It is required to be able to block endogenous subunits (FIG. 1a). This method is necessary because it was suggested that ribosomal subunits containing only synthetic rRNA are shown in a small fraction (up to about 10-15%) of the total cellular 40S ribosomal subunit about 48 hours after transfection. It is.

  There have been no reports of functional studies of nucleotide changes that provide antibiotic resistance to higher eukaryotes. Pactamycin affects translation by binding to rRNA in the E site of the small subunit and preventing the positioning of mRNA at this site. The result of pactamycin binding is the accumulation of dipeptides resulting from the inhibition of extension and the consequence of an aborted translation initiation event. The effects of murine 18S rRNA mutations at the four bases in SEQ ID NO: 23, G963, A964, C1065 and C1066 were analyzed. These bases correspond to positions 693, 694, 795 and 796, respectively, at the E site of E. coli 16S rRNA identified from the crystal structure. Unless otherwise indicated, the numbering of these sites in the mammalian 18S rRNA sequence is based on that of E. coli 16S rRNA. The mutation was introduced into a construct (p18S.1 (Pol-1)) expressing untagged mouse 18S rRNA and containing the full length 5'ETS and ITS1 spacer region. Pactamycin resistance was assessed by 35S-Met / Cys pulse labeling of transfected cells in the presence of 100 ng / ml antibiotic. Purine-purine (G693A, A694G) and pyrimidine-pyrimidine (C795U, C796U) transposition mutations occurred at each nucleotide site thought to bind pactamicin. Cell lysates were compared by SDS-PAGE to untransfected cells and cells expressing synthetic 18S rRNA and synthetic mutant 18S rRNA (FIG. 4a). Mature ribosomes were also transfected with cells transfected with the luciferase reporter plasmid (pGL4.13) in the presence of 100 ng / ml pactamycin and analyzed for their ability to translate luciferase mRNA (FIG. 4b). The results of both experimental sets indicate that all four mutations provide some degree of pactamycin resistance. The G693A and A694G mutations showed the highest level of resistance, while the C795U and C796U mutations gave results slightly above background. The labeling results (FIG. 4a) suggest that none of the mutants significantly changed the protein binding pattern compared to wild type ribosomes.

  The G693A mutation that provided the highest level of pactamycin resistance was used exclusively for further experiments. The level of synthetic 18S rRNA for constructs with each mutation was monitored using hybridization tags and found that there were no substantial differences that could lead to differences in translation between G693A and other mutants ( FIG. 4c). To further characterize the G693A mutation, cells expressing wild type or mutant ribosomes were cultured under increasing concentrations of pactamycin. The results showed that translation from the wild type 40S subunit was substantially inhibited by 100 ng / ml pactamycin but could be further inhibited at higher concentrations. In contrast, translation from mutant subunits was found to be unaffected even when tested at a high concentration of 6,400 ng / ml (FIG. 4d).

  Further evidence of subunit function was obtained from sucrose density analysis of the distribution of ribosomes containing the G693A mutation. In these experiments, the distribution of ribosomal subunits, including wild type (endogenous) and synthetic (G693A) 18S rRNA, was hybridized downstream of ddCTP as a G693A mutation and terminator using oligonucleotide primers (693RT). (Fig. 5a). Under these reaction conditions, the mutant 18S rRNA produces a primer extension product that is 2 nucleotides longer than that generated from the endogenous rRNA. This primer extension reaction allows analysis of the presence and absence of these rRNAs within the same sample. For these experiments, lysates were prepared from N2a cells transfected with G693A construct and treated with cycloheximide or EDTA. The lysate was then fractionated by sucrose density gradient (FIG. 5b, c) and the resulting fractions were analyzed by primer extension (FIG. 5d, e). Analysis of primer extension product distribution by cycloheximide profile (FIG. 5b, d) showed that the mutant rRNA had a relative distribution similar to that of endogenous 18S rRNA throughout the polysome. Quantification of WT and G693A primer extension products and comparison of their relative ratios showed a relatively equal distribution by gradient (in FIG. 5d), where the ribosomal subunit containing the synthetic 18S rRNA is similar to the function of the endogenous subunit It is suggested that it has the function of Similarly, the presence of mutant 18S rRNA in the 40S subunit was confirmed via co-localization with lysate treated with EDTA and the 40S peak (Fig. 5c, e).

Example 5 Based on the functional size of the 40S subunit from the rRNA precursor with the spacer region deletion, several deletion constructs were identified that were thought to yield appropriately processed mature 18S rRNA (Figure 2 and 3). However, cleavage at the correct site ensures that these rRNAs are properly folded and incorporated into functional subunits, as the interaction of the spacer region and 18S rRNA may be required for correct folding. May not be enough. To determine whether rRNAs with deletions in the 5′ETS and ITS1 can adopt a functional structure for the 40S ribosomal subunit, the G693A mutation was constructed in the construct p18S. 7 and. They were tested in cells transfected into each of the two and co-transfected with either the monocistronic luciferase reporter construct (Figure 6a) or the dicistronic double luciferase vector (Figure 6b). Expression in individual cases was measured in the presence of pactamycin to inhibit translation from the endogenous 40S subunit. It is anticipated that structural changes resulting from incorrect ribosome formation or folding in the deletion constructs can have various effects on the translation of these mRNAs, which are cap-dependent translations and different initiations for each translational ability Represents two classes of IRES-dependent translations that require factors.

  For monocistronic constructs, no significant differences were observed between ribosomes, and deletions in the spacer sequence did not disrupt ribosome accumulation or the ability of the subunit to capture translation factors associated with cap-dependent translation. It was suggested that there was no effect (Fig. 6a). For the dicistronic construct, the expression of the second cistron was measured and promoted by either EMCV or PV IRES in the intercistronic region compared to a control sequence that had no known IRES activity. The results show that the expression ratio of the two cistrons (hRen / luc2; FIG. 6b) was similar for the control and spacer-deleted rRNA constructs, and the ribosomal subunit in each case was mediated through these IRES. This suggests that the ability to translate mRNA is indistinguishable from wild type. These results further support the conclusion that ribosomal subunits from short rRNA transcripts are active and do not require sequences deleted from 5'ETS and ITS1.

Example 6 Effect of 5'ETS and ITS1 on the overall efficiency of subunit formation Deletions in 5'ETS and ITS1 do not appear to affect the translational ability of the 40S subunit, but these flanking sequences are It was unclear whether it could affect the production efficiency. The transfection conditions for this experiment were optimized by masking differences in the relative abundance of subunits from individual constructs in order to maximize ribosomal subunit production. Experiments were therefore performed to reduce the expression of synthetic rRNA per cell by cells transfected with diluted plasmid constructs. These experiments were performed using standard transfection conditions with the same amount of total plasmid per transfection. However, the amount of p18S rRNA expression plasmid (1 μg, 0.1 μg, and 0.01 μg) was changed by using another plasmid (pBS KS) as a filler.

  The three expression constructs tested in this experiment contained a full length 5′ETS and ITS1 sequence (p18S.1), a deletion in 5′ETS (p18S.7), and a deletion in ITS1 (p18S.2). Including. All three constructs contain a hybridization tag for detection of mature 18S rRNA. Cells transfected with different plasmids at different dilutions showed no substantial difference when compared by Northern blot (FIG. 9). Taken together, the results indicate that deletion of the adjacent spacer region does not affect translation from the mature subunit or its accumulation. However, this study does not exclude the possibility that adjacent regions can have a slight effect. The results also show that p18S. 1 Shows that logarithmic dilution of the vector does not result in a logarithmic decrease in the level of mature 40S subunit, and that the standard conditions used in the experiment (1.0 μg plasmid) show the ability of cells to express the 40S ribosomal subunit. Suggests saturation.

Example 7 Materials and Methods rDNA cloning and mutagenesis: 18S rDNA and flanking regions were generated from genomic DNA generated from mouse N2a cells and oligonucleotide primers rDNA. 1 and rDNA. Amplified by PCR using 2 (Table 1). Cloning into the plasmid pRL-CMV (Promega) was performed using rDNA. 1 and rDNA. NheI and NotI restriction enzyme recognition sites introduced at the 5 ′ end of 2 were used. All sequencing of PCR products and plasmid constructs was performed using standard Sanger methods. For detection of synthetic 18S rRNA, the 24 nt sequence was cloned into 18S rDNA as a hybridization tag using the SacI site in extension segment 3. Hybridization tags are generated using a random sequence generator (www.faculty.ucr.edu/~mmaduro/random.htm), with minimal predicted self-complementation and oligo calculation (Oligo Calc) (www.basic .northwestern.edu / biotools / OligoCalc.html) and selected based on secondary structure. A construct containing a hybridization tag is manifested by a 'tag' extension, eg, pPol-I (18S-tag).

  A short RNA polymerase (pol-I) promoter element containing a 5 'SalI-box and a minimal pol-I promoter was added to primer Pol-I. 1 and Pol-I. 2 was cloned from the intergenic spacer of the 45S rDNA gene (-169 to +1). Pol-I. BlgII site and Pol-I. The NheI site in 2 was used to replace the BlgII-NheI fragment containing the CMV promoter and chimeric intron and to clone the PCR fragment into pRL-CMV. The pol-I terminator was added to primer Pol-I. 3 and Pol-I. 4 was cloned from a 3S external transcription spacer (3'ETS) of 45S rDNA containing 10 SalI-box pol-I terminators. Primer Pol-I. 3 and the NotI site and primer Pol-I. The MfeI site in 4 was used to clone the PCR fragment into pRL-CMV, replacing the NotI-MfeI fragment containing the SV40 polyA signal.

  Three 5'ETS and two ITS1 fragments were cloned for these experiments. The 5'ETS fragment is the entire 4,014 nt sequence and two shorter fragments extending 1,188 and 703 nucleotides upstream of site 1. Two larger fragments were cloned using NdeI as the 3 'site immediately after 3' of site 1. 4,014 nt sequence from a PCR product containing the pol-I promoter as a primer, rDNA. 3, and rDNA. 4 was used to amplify. The 1,188 nt fragment was used as a primer for rDNA. 4 and rDNA. The PCR product was amplified using 5 and cloned using the NheI site contained within 5'ETS. The 703nt fragment was ligated with oligonucleotide primer rDNA. 1 and rDNA. In a PCR reaction carried out with 2, it was cloned with 18S rDNA. The two ITS1 fragments are the primers rDNA. 6 and rDNA. A 1 kb fragment amplified with 7 and a 70 nt fragment cloned with 18S rDNA. The 1 kb fragment was ligated to Subcloning was performed using the NotI site introduced in 7 and the EcoNI site present at the 3 'end of 18S rDNA. The various fragments described above were used to create six constructs. A construct containing the full length 5'ETS was constructed using the following restriction enzyme fragment combinations. MfeI-BglII-NdeI-EcoNI-NotI-MfeI. Another construct was constructed using the restriction enzyme fragment combination MfeI-BglII-NheI-NdeI-EcoNI-NotI-MfeI.

  For transcription via the RNA polymerase II (pol-II) promoter, the above inserts other than the full-length 5'ETS insert were incorporated into the plasmid pRL-CMV, replacing the NheI-NotI fragment. The full-length 5'ETS insert is first used to introduce oligonucleotide rDNA.DNA to introduce a 5'-terminal NheI site. 12 was re-amplified. The chimeric intron of pRL-CMV was removed and oligonucleotide rDNA. 8 and rDNA. 9 was replaced with the SacI-NheI fragment generated by annealing.

  The deletion construct was prepared by digesting plasmid pPol-I (18S-tag) with the following enzyme group and then religating. PvuII-AfeI (p18S.11), PvuII-ZraI (p18S.10), AvrII-NheI (p18S.9), XhoI-XhoI (p18S.8), and AatII (blunt) -BglI (blunt) (p18S.7) ). p18S. 7 was made using partial digestion with BglI because the plasmid contains an additional site.

  Mutations were made by PCR using complementary 45 nt primers containing the mutation and 5 'and 3' flanking primers located outside the restriction enzyme sites used for cloning. The fragment from each PCR reaction was then subcloned into the expression construct: 18S NcoI-HindIII fragment for antibiotic resistance mutation or 5'ETS AvrII-AatII fragment for 5 'hinge mutation.

  Reporter gene: pGL4.13 (Promega) was used as a monocistron control. A dicistron control was constructed from pGL4.13 by 3 'double insertion of the synthetic firefly luciferase gene luc2. The first fragment, the XbaI-NcoI fragment, contains multiple cloning sites (MCS), an internal ribosome binding site (IRES) derived from encephalomyocarditis virus (EMCV), or a poliovirus (PV) IRES (10) above Derived from the construct. The second fragment was an NcoI-XbaI fragment from phRG-B (Promega) containing the humanized Renilla luciferase gene hRluc.

Analysis of rRNA expression and processing: All constructs in these experiments were tested in transiently transfected neuro 2a (N2a) cells. Cells were seeded in 6-well dishes at 100,000 cells / well and transfected the next day with 1 μg DNA / well and 3 μl Fugene 6 (Roche) (transfection reagent). Approximately 48 hours after transfection, total RNA was extracted using Trizol reagent (Life Technologies). RNA was quantified and 2 μg total RNA and the indicated amount j of rRNA in vitro transcripts were electrophoresed in an agarose gel in a denatured state for Northern blot analysis. For each sample set, the gel contained a single stranded RNA marker (1 μg ssRNA marker; NEB). The position of the band was marked on the membrane after transfer using ethidium bromide staining. For Northern blot analysis, membranes were hybridized with ³² P- or ³³ P-5 ′ labeled oligonucleotide probes in Ultrahyb solution (Ambion) overnight at 37 ° C. The blot was analyzed using a Molecular Dynamics phosphoimager system. Different oligonucleotide probes (Table 1) were used to detect RNA containing different sequences. Α-tag probe for RNA containing 24-nt hybridization tag; α-5′ETS probe for RNA containing 5′ETS sequence immediately upstream of site 1, RNA containing ITS1 sequence immediately after 3 ′ of site 2 [Alpha] -ITS1 probe for use, and [alpha] -18S rRNA probe for 18S rRNA.

  T7 RNA polymerase promoter fused to the first 24 nucleotides of 18S rDNA and a 3 'primer rDNA. Complementary to the nucleotide at the 3' end of 18S rDNA. 5 'primer rDNA. Ambion's MEGAscript in vitro transcription kit was made from PCR-generated fragments amplified using a plasmid template containing 10. The resulting transcript contains three additional 5 'guanine nucleotides compared to the endogenous 18S rRNA. The untagged transcript is 1,874 nucleotides. The tagged transcript is 1,898 nucleotides.

Analysis of synthetic ribosomes: For analysis of protein expression from pactamycin resistant ribosomes, cells were seeded in 24-well dishes at 20,000 cells / well and transfected 24 hours later. 0.5 μg DNA / well was transfected with 1.5 μl Fugene 6 (Roche). Approximately 40 hours after transfection, the cell culture medium was replaced with L-methionine and L-cysteine deficient medium. Cells were starved 30 minutes before the medium was replaced with fresh pactamycin-containing starvation medium as indicated in the figure and incubated for an additional 30 minutes, after which 1.2 μl of TRAN35S label (MP Biomedicals, Ohio) / well was used. And labeled with ³⁵ S-Met / ³⁵ S-Cys for 4 hours. After labeling, the interior was removed and the cells were washed with ice-cold PBS. Cells were treated with 35 μl ice-cold RIPA buffer (10 mM sodium phosphate pH 7.3, 150 mM NaCl, 1 mM EDTA 1% IGEPAL CA-603, 0.1% SDS, 1% sodium deoxycholate, and 1 × complete protease inhibitor ( Roche)) for 30 minutes on ice on a rocker table. Cell lysates were centrifuged at 12,000 xg for 15 minutes to remove debris and pellet genomic DNA. 19 μl of lysate was transferred to a new tube using 1 μl of 1M DTT and 4 × LDS loading buffer (Invitrogen). Samples were heated for 5 minutes at 90 ° C., placed on ice and electrophoresed on a 4-12% BIS-TRIS NuPage gel using MOPS-SDS buffer. Gels were soaked in 30% methanol, soaked in 5% glycerol for 30 minutes, vacuum dried and analyzed using a Molecular Dynamics phosphoimager system or film.

For polysome analysis, cells were seeded in 100,000 wells in 6 well plates and transfected 24 hours later with 18S rDNA expression plasmid and 6 μl Fugene 6 / μg DNA. Samples were processed at 4 ° C. 48 hours after transfection. The culture medium was aspirated and the cells were washed with ice-cold PBS containing 0.1 mg / ml cycloheximide. Six wells were treated with 300 μl of cold lysis buffer A (20 mM Tris-HCl pH 7.5, 15 mM MgCl ₂ , 100 mM KCl, 1% Triton X-100, 0.1 mg / ml cycloheximide, 0.05 × Murine RNase inhibitor (NEB ) 1x complete protease inhibitor (Roche)). The collected cells and lysis buffer were then transferred to tubes and incubated on ice for 10 minutes with occasional mixing for passive lysis. The tube was centrifuged at 16,000 xg for 10 minutes to separate cell debris. Approximately 1 OD ₂₆₀ supernatant was loaded onto a 12 ml 10% -50% sucrose gradient (20 mM Tris-HCl pH 7.5, 15 mM MgCl2, 100 mM KCl, 0.1 mg / ml cycloheximide). The gradient was centrifuged in a SW40Ti rotor at 35,000 rpm (155,000 × g) for 3 hours and then fractionated using an ISCO fractionator. RNA was ethanol precipitated from these fractions and the resulting pellet was extracted with phenol / chloroform. For each collected fraction, 1/10 of the total amount was analyzed by primer extension with ddCTP to distinguish between endogenous rRNA and synthetic rRNA containing pactamycin resistance mutations. Controls for the primer extension reaction included 200 ng 1: 1 18S wt / 18S693 mutant transcript mix (Control 1) and 600 ng N2a total RNA (Control 2). An RNA sequence ladder was generated using 200 ng of the same 1: 1 transcript mix.

For EDTA-separated ribosomes, cells were washed with 1 mL warm PBS 48 hours after transfection and 18 wells were treated with 800 μl ice-cold lysis buffer B (20 mM Tris-HCl pH 7.5, 100 mM KCl, 0.3% Igepal CA-630, 0.05 × Murine RNase inhibitor (NEB), 1 × complete protease inhibitor (Roche)). The lysate was further processed using a dounce homogenizer (100 passes) and then spun for 15 minutes at 16,000 x g to precipitate the residue. The OD ₂₆₀ of each lysate supernatant is determined and 1/5 volume of EDTA buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 150 mM EDTA, 0.05 × Murine RNase inhibitor (NEB), 1 × complete protease inhibition (Roche) was added to adjust the final concentration of EDTA to 30 mM. Each lysate of about 4.5 OD ₂₆₀ was loaded onto a 12 mL 10-35% sucrose gradient in 20 mM Tris-HCl pH 7.5, 100 mM KCl, 30 mM EDTA. The gradient was rotated for 5 hours at 40,000 rpm (200,000 × g) in a SW40Ti rotor. RNA from the fraction samples was analyzed as described above, except that 1/60 of each collected fraction was used for more efficient lysis and enrichment of ribosomal subunits.

P100 pellet and S100 supernatant fractions were prepared using mild lysis conditions to minimize nuclear lysis. Forty-eight hours after transfection, the pellet was washed with 1 ml of warm PBS and then 250 μl of ice-cold lysis buffer B containing 5 mM MgCl ₂ and 2 mM DTT was added to each well. Wells were collected as described above. Cell lysates were actively lysed and centrifuged to precipitate the residue. The supernatant was then centrifuged at 100,000 xg for 3 hours. RNA was isolated from the supernatant as described for the fractions above. P100 pellet, softened overnight with 20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl ₂ and RNA extracted with phenol / chloroform.

  For cotransfection experiments using 18S rDNA and luciferase reporter constructs, cells were seeded in 6-well plates and transfected with 18S rDNA expression constructs using 1 μg vector: 3 μl Fugene 6 / well as described above. For pactamycin treatment, cell medium was removed 48 hours after transfection and replaced with medium containing 100 ng / ml pactamycin. Cells were incubated for about 30 minutes and then transfected with a reporter construct using 1 μg plasmid and 2 μl Lipofectamine 2000 (Invitrogen). Cells were cultured overnight, washed with warm PBS and lysed with passive lysis buffer (Promega). For each independent transfection experiment, an equal volume of cell lysate (20 μl of 250 μl) was assayed using an E & G Berthold 96 well format dual injector luminometer.

  This specification includes many specific examples, which should not be construed as limiting the scope of the invention as claimed or claimed, but rather in a particular embodiment. Describes unique features. Certain features that are described in this specification in the context of separate embodiments can also be provided in combinations of single embodiments. Conversely, the various features described herein in the context of a single embodiment can be provided in multiple embodiments either separately or in any suitable combination. Further, features may be described as implementations of certain combinations and as originally claimed themselves, but one or more features from the claimed combination may be excluded from the combination in certain cases. Well, the claimed combinations may be directed to sub-combinations or variations of sub-combinations. Only a few examples and means are described. Variations, modifications and improvements of the described examples and means as well as other means may be made based on the description herein.

  All references, databases, GenBank sequences, patents, and patent applications mentioned herein are hereby incorporated by reference as if each was specifically and individually indicated to be included by reference. It is.

Claims (25)

  A synthetic or isolated polynucleotide encoding a mammalian 18S rRNA that is resistant to pactamycin (wherein the pactamycin resistance is provided by one or more single base substitutions in the 18S rRNA sequence), the substituent. A fragment thereof containing, a sequence complementary thereto, or a sequence substantially identical thereto.   The polynucleotide according to claim 1, wherein the single-base substitution is at a position corresponding to G963, A964, C1065 or C1066 of mouse 18S rRNA (SEQ ID NO: 23).   The polynucleotide of claim 1 wherein the single base substitution is at a position corresponding to position G963 of SEQ ID NO: 23.   The polynucleotide of claim 1 wherein the single base substitution corresponds to the G963A substitution of SEQ ID NO: 23.   The polynucleotide of claim 1 wherein the polynucleotide encodes human 18S rRNA or mouse 18S rRNA.   The polynucleotide of claim 1 wherein the polynucleotide comprises SEQ ID NO: 23, which does not have a G963A substitution.   A mammalian 18S rRNA encoded by the polynucleotide of claim 1.   An expression vector for expressing the polynucleotide according to claim 1.   9. The vector of claim 8, wherein the polynucleotide further comprises 5'ETS and ITS1 of rDNA sequences.   9. The vector of claim 8, further comprising a pol-I promoter or a cytomegalovirus (CMV) promoter.   9. The vector of claim 8, further comprising a 3'ETS or SV40 poly (A) signal.   A host cell comprising the expression vector according to claim 8.   A kit comprising the expression vector according to any one of claims 8 to 11 and / or a cell containing the expression vector. A method for preferential translation of recombinant mRNA,
The synthetic polynucleotide of claim 1 wherein the synthetic polynucleotide has been modified to introduce one or more mutations that result in preferential binding of 18S rRNA to recombinant mRNA in mammalian cells. To express)
Recombinant mRNA is provided to the cell and the cell is exposed to a sufficient amount of pactamycin to reduce or eliminate protein synthesis from the cell's endogenous 40S ribosomal subunit, and thus protein synthesis in the cell. And preferentially translating the recombinant mRNA, wherein the method is limited to 40S ribosomal subunits comprising 18S rRNA expressed primarily by the synthetic polynucleotide of claim 1. A method for identifying a mutation in 18S rRNA that alters ribosome function comprising:
(A) Additional mutations are introduced into synthetic polynucleotides encoding mammalian 18S rRNA that are resistant to pactamycin (wherein the pactamycin resistance is caused by one or more single base substitutions in the 18S rRNA sequence). ),
(B) expressing in the host cell a synthetic polynucleotide having the further mutation in the presence of pactamycin; and
(C) detecting a change in the ribosome of the host cell compared to a control cell expressing the synthetic polynucleotide without the further mutation, and thus identifying the further mutation as one altered ribosome function. Including a method.   16. The method of claim 15, wherein the synthetic polynucleotide is present in an expression vector introduced into the host cell.   16. The method of claim 15, wherein the pactamycin resistant 18S rRNA has a single base substitution at a position corresponding to G963, A964, C1065 or C1066 of mouse 18S rRNA (SEQ ID NO: 23).   16. The method of claim 15, wherein the single base substitution corresponds to a G963A substitution in SEQ ID NO: 23.   16. The method of claim 15, wherein the synthetic polynucleotide comprises SEQ ID NO: 23 without the G963A substitution. A method for producing a ribosome having enhanced translation efficiency in a mammalian cell, comprising:
(A) Additional mutations are introduced into the synthetic polynucleotide encoding the pactamicin resistant mammalian 18S rRNA (wherein the pactamicin resistance is brought about by one or more single base substitutions in the 18S rRNA sequence).
(B) expressing a synthetic polynucleotide having the additional mutation in a mammalian host cell;
(C) culturing the cells in the presence of pactamycin; and
(D) detecting an enhanced translation efficiency in the cell compared to a control cell expressing the synthetic polynucleotide without said further mutation, and therefore producing a ribosome having an enhanced translation efficiency. Including.   21. The method of claim 20, wherein the synthetic polynucleotide is present in an expression vector introduced into the host cell.   21. The method of claim 20, wherein the pactamycin resistant 18S rRNA has a single base substitution at a position corresponding to G963, A964, C1065 or C1066 of mouse 18S rRNA (SEQ ID NO: 23).   21. The method of claim 20, wherein the single base substitution corresponds to a G963A substitution in SEQ ID NO: 23.   21. The method of claim 20, wherein the synthetic polynucleotide comprises SEQ ID NO: 23 without the G963A substitution.   21. The method of claim 20, wherein translation efficiency is determined by measuring the level of a particular polypeptide in the host cell.

Images